Staple peptides and uses thereof

Abstract

A series of stapled peptides and uses thereof are disclosed, specifically, polypeptides having sequences represented by formulas (I-1) to (I-5) and (II-6) and pharma- ceutically acceptable salts thereof.

Description

[Technical Field]

[0001] Citation of Related Applications This application claims priority to and is entitled to Chinese Invention Patent Application No. 202111398470.4, filed with the State Intellectual Property Office of China on November 19, 2021, and Chinese Invention Patent Application No. 202111500873.5, filed with the State Intellectual Property Office of China on December 9, 2021, the entire disclosures of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a series of stapled peptides and uses thereof, specifically to polypeptides having the sequences shown in formulas (I-1) to (I-5) and (II-6) and pharmaceutically acceptable salts thereof. [Background technology]

[0003] Diabetes mellitus (Diabetes mellitus) is a group of metabolic disorders characterized by hyperglycemia caused by defective insulin secretion, impaired insulin function, or both. Long-term hyperglycemia can cause chronic damage and dysfunction in various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves. According to the latest data from the International Diabetes Federation (2019), there are approximately 460 million people with diabetes worldwide. In 2020, China's diabetic population approached 130 million, making it the country with the highest number of diabetes cases in the world. A nationwide cross-sectional study from China published in the British Medical Journal in 2020 found that the national prevalence of type 2 diabetes (T2D) was 11.2%, while only 49.4% of patients achieved target glycated hemoglobin levels. T2D is a progressive disease, and in the later stages of the disease, patients are at high risk for complications such as diabetic nephropathy, diabetic retinopathy, and diabetic foot. Therefore, T2D is a major health challenge facing humanity.

[0004] Insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are both incretins that promote insulin release and are glucose-dependent, resulting in a low risk of hypoglycemia. GLP-1 inhibits appetite in the brain, delays gastric emptying, reduces hepatic gluconeogenesis, and increases glucose uptake in muscle tissue. GIP increases glucose uptake in adipose tissue, promotes lipid metabolism, and prevents ectopic fat accumulation. Glucagon (GCG), secreted from pancreatic islet alpha cells, inhibits hepatic lipogenesis and cholesterol levels, increases the fat burning rate in brown adipose tissue, and protects cardiomyocytes and the heart. Therefore, triple agonism of GIPR, GLP-1R, and GCGR may have a synergistic effect on the regulation of glucose and lipid metabolism, potentially resulting in more effective reductions in blood glucose levels, weight loss, reduction of fatty liver, and protection against cardiovascular disease. Summary of the Invention

[0005] The present invention provides a polypeptide having the sequence shown in the formula below, or a pharmaceutically acceptable salt thereof. Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp-Lys- 1 Lys-Ala-Gln- 1 Lys-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(I-1), Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp- 1 Lys-Lys-Ala- 1 Lys-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(I-2), Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp-Lys-Lys-Ala-Gln- 1 Lys-Ala-Phe- 1 Lys-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(I-3), Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp-Lys-Lys-Ala-Gln-Aib- 1 Lys-Phe-Ile- 1 Lys-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(I-4), Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp-Lys-Lys-Ala-Gln-Aib-Ala-Phe-Ile-Glu- 1 Lys-Leu-Leu- 1 Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(I-5), Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X1-Leu-Asp-Lys-Lys-Ala-Gln-Aib-Ala-Phe-Ile- 1 Lys-Tyr-Leu- 1 Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X2(II-6), However, the structure of Aib is

Chem.

Chem.

[0006] In some embodiments of the present invention, m is selected from 2, and the other variables are as defined herein.

[0007] In some embodiments of the invention, n is selected from 9, and other variables are as defined herein.

[0008] In some embodiments of the present invention, p is selected from 1, and the other variables are as defined herein.

[0009] In some embodiments of the invention, X2 is [ka] and the other variables are as defined in the present invention.

[0010] In some embodiments of the present invention, X0 is [ka] and the other variables are as defined in the present invention.

[0011] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined in the present invention.

[0012] The present invention further provides a polypeptide represented by the following formula or a pharmaceutically acceptable salt thereof: [ka] [ka] [ka] [ka] [ka] [ka]

[0013] The present invention further provides a pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the above polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0014] One aspect of the present invention is the use of the above-described polypeptide or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition in the manufacture of a medicament for treating diabetes and / or obesity. [Effects of the Invention]

[0015] The polypeptides of the present invention have strong agonistic activity against GLP-1R / GIPR, have excellent pharmacokinetic properties in multiple species, exhibit excellent weight loss effects in DIO mice, and exhibit excellent blood glucose lowering effects in db / db mice.

[0016] (Definitions and Explanations) Unless otherwise stated, the following terms and phrases used herein have the following meanings. Unless otherwise defined, a particular term or phrase should be understood to have its ordinary definition, rather than being indefinite or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0017] As used herein, "pharmaceutically acceptable" refers to polypeptides, materials, compositions and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals and are consistent with a reasonable benefit / risk ratio without significant toxicity, irritation, allergic response or other problem or complication.

[0018] The term "pharmaceutically acceptable salt" refers to a salt of a polypeptide of the present invention, which is prepared with a relatively non-toxic acid or base when the polypeptide has specific substituents discovered in the present invention. When the polypeptide of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the polypeptide with a sufficient amount of base in a solution alone or in a suitable inert solvent. Some specific polypeptides of the present invention contain basic and acidic functional groups and can therefore be converted into any base addition salt or acid addition salt.

[0019] The pharmaceutically acceptable salts of the present invention can be synthesized from parent polypeptides containing acidic or basic groups by conventional chemical methods. Typically, such salts are prepared by reacting the free acid or base form of the polypeptide with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both.

[0020] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that perform functions similar to those of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that have subsequently been modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are polypeptides that have the same basic chemical structure as a naturally occurring amino acid (e.g., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds whose structure differs from the general chemical structure of an amino acid but perform a similar function to a naturally occurring amino acid.

[0021] As used herein, A or Ala represents alanine and the structure is [ka] where R or Arg represents arginine and the structure is [ka] wherein N or Asn represents asparagine and the structure is [ka] where D or Asp represents aspartic acid and the structure is [ka] where C or Cys represents cysteine ​​and the structure is [ka] where Q or Gln represents glutamine and the structure is [ka] where E or Glu represents glutamic acid and the structure is [ka] where G or Gly represents glycine and the structure is [ka] where H or His represents histidine and the structure is [ka] where I or Ile represents isoleucine and the structure is [ka] where L or Leu represents leucine and the structure is [ka] where K or Lys represents lysine and the structure is [ka] where M or Met represents methionine and the structure is [ka] where F or Phe represents phenylalanine and the structure is [ka] where P or Pro represents proline and the structure is [ka] where S or Ser represents serine and the structure is [ka] where T or Thr represents threonine and the structure is [ka] where W or Trp represents tryptophan and the structure is [ka] wherein Y or Tyr represents tyrosine and the structure is [ka] wherein V or Val represents valine and the structure is [ka] is.

[0022] The term "treatment" includes inhibiting, alleviating, halting or reversing the progression or severity of an existing condition or disease.

[0023] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, enantiomers and tautomers.

[0024] Polypeptides of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such polypeptides, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof as well as other mixtures, such as mixtures enriched in enantiomers or diastereomers, and all such mixtures are within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers and mixtures thereof are within the scope of the present invention.

[0025] Unless otherwise specified, the terms "enantiomers" or "optical isomers" are stereoisomers that are mirror images of each other.

[0026] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" refer to the inability to freely rotate about double bonds or single bonds of ring carbon atoms.

[0027] Unless otherwise specified, the term "diastereomer" refers to stereoisomers whose molecules have two or more centers of chirality and whose molecules are not mirror-images of each other.

[0028] Unless otherwise stated, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic. [ka]

[0029] Unless otherwise explained, the terms "enriched in one isomer," "isomer-enriched," "enriched in one enantiomer," or "enantiomer-enriched" mean that the amount of one isomer or enantiomer is less than 100% and that the amount of that isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0030] Unless otherwise stated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomeric or enantiomeric excess (ee) is 80%.

[0031] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared using asymmetric synthesis, chiral reagents, or other conventional techniques. Single enantiomers of certain compounds of the invention can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary is cleaved to provide the desired isolated enantiomer. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxyl) functional group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by separation of the diastereomers by conventional methods known in the art and subsequent recovery to provide the isolated enantiomers. Separation of enantiomers and diastereomers is also typically accomplished by chromatographic methods using chiral stationary phases and optionally chemical derivatization (e.g., carbamate formation from an amine).

[0032] The compounds of the present invention may contain unnatural atomic isotopes at one or more atoms constituting the compounds. For example, tritium ( 3 H), iodine-125( 125 I) or C-14( 14 Compounds can be labeled with radioactive isotopes such as C). For example, deuterium can be substituted for hydrogen to form deuterated drugs, where the bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs offer the advantages of reduced toxic side effects, increased drug stability, improved therapeutic efficacy, and a longer biological half-life. Conversion of the isotopic composition of the compounds of the present invention, whether radioactive or not, is within the scope of the present invention.

[0033] If the listed linking group does not specify any other linking direction, the linking direction is arbitrary, for example: [ka] In the formula, the linking group L is -MW-, and in this case, -MW- constitutes ring A and ring B in the same direction as the reading order from left to right. [ka] You can also construct rings A and B in the reverse order of reading from left to right. [ka] Combinations of the above linking groups, substituents and / or variables are permissible only if such combinations result in stable compounds.

[0034] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups by chemical bonds. If the bonding mode of the chemical bond is delocalized and there is an H atom at the bondable site, when a chemical bond is formed, the number of H atoms at the site is reduced to the corresponding valence of the group according to the number of bonded chemical bonds. The chemical bond that bonds the site to another group is represented by a straight solid bond ( [ka] ), straight dashed bond ( [ka] ), or wavy line ( [ka] For example, the straight solid bond in -OCH3 means that the group is bonded to another group via its oxygen atom. [ka] A straight dashed bond within a group means that both ends of the nitrogen atom within the group are bonded to other groups. [ka]

[0035] The wavy line in the middle means that the phenyl group is bonded to another group via carbon atoms at positions 1 and 2 of the phenyl group.

[0036] Unless otherwise defined, the term "C 1-3 "Alkyl" refers to a saturated hydrocarbon group consisting of 1 to 3 carbon atoms, either straight or branched. 1-3 C for alkyl 1-2 and C 2-3 alkyl, which may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). 1-3 Illustrative examples of alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl).

[0037] The structure of the polypeptide of the present invention can be confirmed by conventional methods known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means known to those skilled in the art. For example, single crystal X-ray diffraction (SXRD), the cultured single crystal is collected by a Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting the relevant data, the absolute configuration can be confirmed by direct crystal structure analysis (Shelxs97).

[0038] The polypeptides of the present invention can be produced by a variety of synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthesis methods, and equivalent alternative methods familiar to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0039] All solvents used in this invention are commercially available.

[0040] The present invention uses the following abbreviations: aq represents water; eq represents equivalent; DCM represents dichloromethane; PE represents petroleum ether; DMSO represents dimethyl sulfoxide; MeOH represents methanol; Boc represents tert-butoxycarbonyl, an amine protecting group; Dde represents (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, an amino acid side chain protecting group; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; BocO represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide. HBTU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT represents 1-hydroxybenzotriazole; HOAT represents 1-hydroxy-7-azabenzotriazole; DIC represents N,N'-diisopropylcarbodiimide; DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene; PhSiH3 represents phenylsilane; Pd(PPh3)4 represents tetrakis(triphenylphosphine)palladium; AEEA represents 2-(2-(2-aminoethoxy)ethoxy)acetic acid; and DIEA represents diisopropylethylamine.

[0041] Compounds are named according to conventional naming principles in the art or ChemDraw® software; commercially available compounds use the manufacturer's catalogue name. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will be specifically described below by way of examples, but is not intended to be an adverse limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0043] Intermediate 1 [ka]

[0044] 1. Hanging resin 1.1 20.0 g of chloro(o-chlorophenyl)diphenylmethane (2-CTC Resin (degree of substitution S = 1.00 mmol / g)) and 7.72 g of Fmoc-AEEA-OH were weighed and added to a reaction column, followed by the addition of DCM (40 mL). Next, 14.0 mL of DIEA was added to the reaction column and nitrogen gas was bubbled through for 2 hours. 20.0 mL of MeOH was added to the reaction column and nitrogen gas was continued to be bubbled through for 30 minutes. The waste liquid was discharged until no more liquid came out. DMF (300 mL) was added and washed five times for 1 minute each time, and the waste liquid was discharged until no more liquid came out.

[0045] 1.2 20% piperidine / DMF (300 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (100 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Ninhydrin detection was performed, and the resin was blue.

[0046] 2. Amino acid coupling 2.1 Coupling of Fmoc-AEEA-OH 1. Weigh out Fmoc-AEEA-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 120 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 2. After reacting for 20 minutes at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF for 1 minute each, and the waste liquid was drained until no more liquid came out.

[0047] 2.2 Coupling of Fmoc-Glu-OtBu 1. 20% piperidine / DMF (300 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. Ninhydrin detection was performed and the resin was blue. 2. Weigh out Fmoc-Glu-OtBu (3.0 eq) and add it to the resin. Add DIEA (3.00 eq). Add 300 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (300 mL) for 1 minute each, and the waste liquid was drained until no more liquid came out.

[0048] 2.3 Coupling of 20-(tert-butoxy)-20-oxoicosanoic acid 1. 20% piperidine / DMF (300 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (300 mL) was added and washed five times for 1 minute each time. The waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. 20-(tert-butoxy)-20-oxoicosanoic acid (3.0 eq) was weighed and added to the resin, followed by DIEA (6.00 eq). 300 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid had dissolved, HBTU (2.85 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF for 1 minute each, and the waste liquid was drained until no liquid came out.

[0049] 3. Cleavage and Drying of Crude Peptide 3.1. Cutting fluid was prepared in the following volumes: 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) / DCM=20 / 80.

[0050] 3.2. 400 mL of the prepared cutting solution was poured into a reactor containing the dried peptide resin, bubbled in the reactor for 20 minutes, filtered, and the filtrate was added to the flask. This procedure was repeated twice, and the cutting solution collected in both times was spin-dried to obtain crude peptide intermediate 1.

[0051] The various types of polypeptides included in the examples of the present invention are synthesized according to standard solid phase synthesis methods and purified by reverse phase HPLC.

[0052] Example 1 [ka]

[0053] 1. Weigh out 1.78 g of 4-(2',4'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamide-methyldiphenylmethylamine resin (Rink Amide MBHA Resin (degree of substitution Sub = 0.28 mmol / g)) and add it to a reaction column. Then, add DMF (50 mL) to the reaction column and bubble with nitrogen gas for 2 hours. Discharge the waste liquid until no more liquid comes out. Wash with DMF (50 mL) five times for 1 minute each time and discharge the waste liquid until no more liquid comes out.

[0054] 2. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue.

[0055] 3. Amino acid coupling 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out.

[0056] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0057] 3.3 Fmoc-Pro-OH Coupling 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF for 1 minute each, and the waste liquid was drained until no liquid came out.

[0058] 3.4 Fmoc-Pro-OH Coupling 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0059] 3.5 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0060] 3.6 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0061] 3.7 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0062] 3.8 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0063] 3.9 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0064] 3.10 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0065] 3.11 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0066] 3.12 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0067] 3.13 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Leu-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0068] 3.14 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Leu-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0069] 3.15 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Tyr(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0070] 3.16 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0071] 3.17 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ile-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0072] 3.18 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Phe-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0073] 3.19 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0074] 3.20 Coupling of Fmoc-Lys(Dde)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Lys(Dde)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0075] 3.21 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gln(Trt)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0076] 3.22 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0077] 3.23 Coupling of Fmoc-Lys(Alloc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Lys(Alloc)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0078] 3.24 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Lys(Boc)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0079] 3.25 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Asp(OtBu)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 2 hours at 25°C, ninhydrin detection was performed and the resin was blue. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0080] 3.26 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Leu-OH and add it to the resin. 6.00 eq of HOAT was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid and HOAT were dissolved, DIC (6.00 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting at 25°C for 1 hour, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0081] 3.27 Coupling of Fmoc-α-Me-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-α-Me-Leu-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0082] 3.28 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ile-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble nitrogen gas through the column. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow rate so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0083] 3.29 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0084] 3.30 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Tyr(tBu)-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0085] 3.31 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0086] 3.32 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Ser(tBu)-OH and add it to the resin. 6.00 eq of HOBT was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid and HOBT were dissolved, 6.00 eq of DIC was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0087] 3.33 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Thr(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After overnight reaction at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0088] 3.34 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Phe-OH and add it to the resin. Then, add 6.00 eq of HOBT. 10 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid and HOBT were dissolved, DIC (6.00 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0089] 3.35 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Thr(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After overnight reaction at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0090] 3.36 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Boc-His(Trt)-OH (6.0 eq) was weighed and added to the resin, followed by DIEA (12.0 eq). 10 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid had dissolved, HATU (5.70 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0091] 3.37 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Gln(Trt)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0092] 3.38 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Aib-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0093] 3.39 Coupling of Boc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Boc-Tyr(tBu)-OH (6.0 eq) was weighed and added to the resin, followed by DIEA (12.0 eq). 10 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid had dissolved, HATU (5.70 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0094] 3.40 Deprotection of Alloc 1. PhSiH3 (10.0 eq) and DCM (10 mL) were added to a reaction column and bubbled with nitrogen gas. Pd(PPh3)4 (0.1 eq) was then added and bubbled with nitrogen gas for 20 minutes. This reaction was repeated twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF five times (50 mL each time) for 1 minute, then drain the waste liquid until no more liquid came out.

[0095] 3.41 Coupling of Fmoc-Ida-OH 1. Weigh out 6.0 eq of Fmoc-Ida-OH (CAS#: 112918-82-8) and add it to the resin. Add 12.0 eq of DIEA, add 10 mL of DMF to the reaction column, and bubble with nitrogen gas. After the amino acid was dissolved, add 5.70 eq of HBTU. Adjust the nitrogen gas flow so that the resin swells evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out.

[0096] 3.42 Coupling of Intermediate 1 1. 10% DBU / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 1.50 eq of intermediate 1 and add it to the resin. Then, add 3.00 eq of DIEA and 10 mL of DMF to the reaction column. Bubble nitrogen gas through the column. After the amino acid has dissolved, add 1.45 eq of HBTU. Adjust the nitrogen gas flow rate so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0097] 3.43 Deprotection of Dde 1. 3% hydrazine hydrate / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 15 minutes. The waste liquid was then drained, and the column was washed five times with 50 mL of DMF for 1 minute each. The waste liquid was then drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue.

[0098] 3.44 Amide Ring Closure 1. DIEA (3.0 eq) was added to the DMF solution of the resin, and then HATU (1.5 eq) dissolved in DMF was slowly added dropwise to the reaction column, followed by bubbling with nitrogen gas. The nitrogen gas flow was adjusted so that the resin swelled evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 minutes each, the waste liquid was drained until no more liquid came out, and the resin was poured out, dried and stored.

[0099] 4. Cleavage and Drying of Crude Peptide 4.1 Cutting fluid was prepared in the following volumes: TFA / Tis / H2O / Mpr=90 / 2.5 / 2.5 / 5. 4.2 The dried peptide resin was added to the prepared cutting fluid and shaken on a shaker for 2.5 hours, then filtered. The filtrate was added to 10 volumes of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 hours and purified to obtain WX001. The molecular weight of the polypeptide was confirmed by ESI-MS: the calculated value [M+3H] / 3 was 1673.2, and the detected value was 1673.0.

[0100] Example 2 [ka]

[0101] Polypeptide WX002 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value [M+3H] / 3 of 1658.9 and a detected value of 1658.7.

[0102] Example 3 [ka]

[0103] Polypeptide WX003 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value [M+3H] / 3 of 1678.2 and a detected value of 1678.1.

[0104] Example 4 [ka]

[0105] Polypeptide WX004 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+3H] / 3 was 1677.6, and the detected value was 1677.6.

[0106] Example 5 [ka]

[0107] 1. 2 g of 4-(2',4'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamide-methyldiphenylmethylamine resin (Rink Amide MBHA Resin (degree of substitution Sub = 0.28 mmol / g)) was weighed and added to a reaction column. DMF (50 mL) was then added to the reaction column, and nitrogen gas was bubbled through the column for 2 hours. The waste liquid was then discharged until no more liquid came out. DMF (50 mL) was added and the column was washed five times, each time for 1 minute, and the waste liquid was then discharged until no more liquid came out.

[0108] 2. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue.

[0109] 3. Amino acid coupling 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out.

[0110] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0111] 3.3 Fmoc-Pro-OH Coupling 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF for 1 minute each, and the waste liquid was drained until no liquid came out.

[0112] 3.4 Fmoc-Pro-OH Coupling 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0113] 3.5 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0114] 3.6 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0115] 3.7 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0116] 3.8 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0117] 3.9 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0118] 3.10 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0119] 3.11 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0120] 3.12 Coupling of Fmoc-Lys(Dde)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Lys(Dde)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0121] 3.13 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Leu-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0122] 3.14 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Leu-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0123] 3.15 Coupling of Fmoc-Lys(Alloc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Lys(Alloc)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0124] 3.16 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0125] 3.17 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ile-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0126] 3.18 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Phe-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0127] 3.19 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0128] 3.20 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Aib-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0129] 3.21 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Gln(Trt)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0130] 3.22 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0131] 3.23 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Lys(Boc)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0132] 3.24 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Lys(Boc)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0133] 3.25 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Asp(OtBu)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 2 hours at 25°C, ninhydrin detection was performed and the resin was blue. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0134] 3.26 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Leu-OH and add it to the resin. 6.00 eq of HOAT was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid and HOAT were dissolved, DIC (6.00 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting at 25°C for 1 hour, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0135] 3.27 Coupling of Fmoc-α-Me-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-α-Me-Leu-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0136] 3.28 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ile-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble nitrogen gas through the column. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow rate so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0137] 3.29 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Ser(tBu)-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0138] 3.30 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Tyr(tBu)-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HATU (5.70 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0139] 3.31 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0140] 3.32 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Ser(tBu)-OH and add it to the resin. 6.00 eq of HOBT was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid and HOBT were dissolved, 6.00 eq of DIC was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0141] 3.33 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Thr(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After overnight reaction at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0142] 3.34 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Phe-OH and add it to the resin. Then, add 6.00 eq of HOBT. 10 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid and HOBT were dissolved, DIC (6.00 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0143] 3.35 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out Fmoc-Thr(tBu)-OH (3.0 eq) and add it to the resin. Add DIEA (6.00 eq). Add 10 mL of DMF to the reaction column, bubble with nitrogen gas, and after the amino acid has dissolved, add HATU (2.85 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 3. After overnight reaction at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0144] 3.36 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Boc-His(Trt)-OH and add it to the resin. Add 12.0 eq of DIEA, add 10 mL of DMF to the reaction column, and bubble with nitrogen gas. After the amino acid was dissolved, add 5.70 eq of HATU. Adjust the nitrogen gas flow rate so that the resin swells evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0145] 3.37 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Gln(Trt)-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0146] 3.38 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 6.0 eq of Fmoc-Aib-OH and add it to the resin. 12.0 eq of DIEA was added, and 10 mL of DMF was added to the reaction column. Nitrogen gas was bubbled through the column. After the amino acid dissolved, 5.70 eq of HATU was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0147] 3.39 Coupling of Boc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no liquid came out. Chloranil detection was performed and the resin was green. 2. Boc-Tyr(tBu)-OH (6.0 eq) was weighed and added to the resin, followed by DIEA (12.0 eq). 10 mL of DMF was added to the reaction column, and nitrogen gas was bubbled through. After the amino acid had dissolved, HATU (5.70 eq) was added. The nitrogen gas flow was adjusted so that the resin swelled evenly. 3. After reacting for 0.5 hours at 25°C, chloranil detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0148] 3.40 Deprotection of Alloc 1. PhSiH3 (10.0 eq) and DCM (10 mL) were added to a reaction column and bubbled with nitrogen gas. Pd(PPh3)4 (0.1 eq) was then added and bubbled with nitrogen gas for 20 minutes. This reaction was repeated twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF five times (50 mL each time) for 1 minute, then drain the waste liquid until no more liquid came out.

[0149] 3.41 Coupling of Fmoc-Ida-OH 1. Weigh out Fmoc-Ida-OH (6.0 eq) and add it to the resin. Add DIEA (12.0 eq). Add 10 mL of DMF to the reaction column. Bubble with nitrogen gas. After the amino acid has dissolved, add HBTU (5.70 eq). Adjust the nitrogen gas flow so that the resin swells evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out.

[0150] 3.42 Coupling of Intermediate 1 1. 10% DBU / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 20 minutes. The waste liquid was drained until no more liquid came out. DMF (50 mL) was added and washed five times for 1 minute each, and the waste liquid was drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue. 2. Weigh out 1.50 eq of intermediate 1 and add it to the resin. Then, add 3.00 eq of DIEA and 10 mL of DMF to the reaction column. Bubble nitrogen gas through the column. After the amino acid has dissolved, add 1.45 eq of HBTU. Adjust the nitrogen gas flow rate so that the resin expands evenly. 3. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 4. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no more liquid came out.

[0151] 3.43 Deprotection of Dde 1. 3% hydrazine hydrate / DMF (50 mL) was added to the reaction column, and nitrogen gas was bubbled through for 15 minutes. The waste liquid was then drained, and the column was washed five times with 50 mL of DMF for 1 minute each. The waste liquid was then drained until no more liquid came out. Ninhydrin detection was performed, and the resin was blue.

[0152] 3.44 Amide Ring Closure 1. DIEA (3.0 eq) was added to the DMF solution of the resin, and then HATU (1.5 eq) dissolved in DMF was slowly added dropwise to the reaction column, followed by bubbling with nitrogen gas. The nitrogen gas flow was adjusted so that the resin swelled evenly. 2. After reacting for 0.5 hours at 25°C, ninhydrin detection was performed and the resin was colorless and transparent. 3. The reaction solution was removed by suction, and the column was washed five times with DMF (50 mL each time) for 1 minute, and the waste liquid was drained until no liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 minutes each, the waste liquid was drained until no more liquid came out, and the resin was poured out, dried and stored.

[0153] 4. Cleavage and Drying of Crude Peptide 4.1 Cutting fluid was prepared in the following volumes: TFA / Tis / H2O / Mpr=90 / 2.5 / 2.5 / 5. The dried peptide resin was added to the prepared cutting fluid and shaken on a shaker for 2.5 hours, then filtered. The filtrate was added to 10 volumes of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. After 2 hours of vacuum drying, the crude peptide was obtained and purified to obtain polypeptide WX005. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated [M+3H] / 3 of 1646.9 and a detected value of 1646.9.

[0154] Example 6 [ka]

[0155] Polypeptide WX006 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value [M+3H] / 3 of 1663.5 and a detected value of 1663.3.

[0156] Biological Test Data Experimental Example 1: In vitro GLP-1R / GIPR / GCGR agonist activity test A: Main materials 1) Cell line The cell line was constructed by Shanghai WuXi AppTec New Pharmaceutical Development Co., Ltd. The details are shown in Table 1. [Table 1]

[0157] 2) Reagents and consumables are as shown in Table 2: [Table 2]

[0158] 3) The equipment is as shown in Table 3: [Table 3]

[0159] B. Method I) Experimental materials The experimental buffers are as shown in Table 4: [Table 4]

[0160] The preparation of the detection reagent is as shown in Table 5: [Table 5]

[0161] II) Experimental Method a) Preparation of compound plates: Test compounds were diluted 4-fold in 10-point increments, with a starting concentration of 30 μM, and dilutions were completed in Bravo.

[0162] b) Compound transfer: 1) 100 nL of compound was transferred to an OptiPlate-384 plate using an Echo. 2) The OptiPlate-384 plate was centrifuged at 1000 rpm for 5 seconds.

[0163] c) Preparation of cell suspension 1) One GLP-1R / GIPR / GCGR cell freezing tube was placed in 37°C warm water and allowed to thaw quickly. 2) The cell suspension was transferred to a 15 mL centrifuge tube and gently rinsed with 10 mL of HBSS. 3) The tube was centrifuged at 1000 rpm at room temperature for 1 minute. 4) The supernatant was discarded. 5) The cells at the bottom were gently dispersed, gently rinsed with 10 mL of HBSS, centrifuged to sediment the cells, and finally resuspended in the test buffer. 6) Cell density and viability were measured using Vi-cell. 7) GLP-1R / GIPR / GCGR cell concentration was increased to 2.0 x 10 in test buffer. 5 / mL. 8) 100 nL of the diluted cell suspension was placed in an OptiPlate-384 plate. 9) Incubated at room temperature for 30 minutes.

[0164] d) Addition of detection reagent: 1) 10 μL of 800 nM gradient diluted cAMP standard was added to blank wells of an OptiPlate-384 plate. 2) 10 μL of cAMP detection reagent was added. 3) The OptiPlate-384 plate was covered with TopSeal-A film and incubated at room temperature for 60 minutes. TopSeal-A was removed and the data was read using EnVision.

[0165] C. The experimental results are shown in Table 6. [Table 6]

[0166] Conclusion: The polypeptides of the present invention have strong agonistic activity against GLP-1R / GIPR. Among them, different peptides GCGR These three targets have significant differences in their agonistic activity, which provides an important guideline for studying the balance of activity among the three targets.

[0167] Experimental Example 2: Pharmacokinetic evaluation in rats A. Experimental Objective The purpose was to test the pharmacokinetics of the polypeptide of the present invention in the body of SD rats.

[0168] B. Experimental Procedure The pharmacokinetic properties of the polypeptide of the present invention were tested in rodents after subcutaneous injection using standard protocols. In the experiment, the polypeptide was prepared into a clear solution and then administered to rats via a single subcutaneous injection (SC, 0.048 mpk). The injection solvent was citrate buffer (20 mM, pH = 7). Whole blood was collected to prepare plasma, and the drug concentration was analyzed by LC-MS / MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0169] C. Experimental Results The experimental results are shown in Table 7. [Table 7]

[0170] Conclusion: The polypeptide of the present invention has excellent pharmacokinetic properties in rats.

[0171] Experimental Example 3: Pharmacokinetic evaluation in mice A. Experimental Objective The purpose was to test the pharmacokinetics of the polypeptide of the present invention in C57BL / 6 mice.

[0172] B. Experimental Procedure The pharmacokinetic properties of the polypeptide of the present invention were tested in rodents after intravenous and subcutaneous injection using standard protocols. In the experiment, the polypeptide was prepared into a clear solution and then administered to mice via a single subcutaneous injection (SC, 0.048 mpk). The subcutaneous injection solvent was citrate buffer (20 mM, pH = 7). Whole blood was collected and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0173] C. Experimental Results The experimental results are shown in Table 8. [Table 8]

[0174] Conclusion: The polypeptide of the present invention has excellent pharmacokinetic properties in mice.

[0175] Experimental Example 4: Pharmacokinetic evaluation in cynomolgus monkeys A. Experimental Objective The purpose was to test the pharmacokinetics of the polypeptide of the present invention in cynomolgus monkeys.

[0176] B. Experimental Procedure The pharmacokinetic properties of the polypeptide of the present invention in mammals after intravenous and subcutaneous injection were tested using standard protocols. In the experiment, the polypeptide was prepared into a clear solution and then administered to cynomolgus monkeys via a single subcutaneous injection (SC, 0.02 mpk). The subcutaneous injection solvent was citrate buffer (20 mM, pH = 7). Whole blood was collected to prepare plasma, and the drug concentration was analyzed by LC-MS / MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0177] C. Experimental Results The experimental results are shown in Table 9. [Table 9]

[0178] Conclusion: The polypeptide of the present invention has excellent pharmacokinetic properties in monkeys.

[0179] Experimental Example 5: Drug efficacy study in DIO mice - In vivo drug efficacy evaluation A. Experimental Objective The purpose is to study the weight loss effect of the polypeptide of the present invention in DIO mice.

[0180] B. Experimental Procedure After arriving at the WuXi AppTec facility, DIO mice were housed in a strictly environmentally controlled animal room maintained at a temperature of 20–24°C and humidity of 30–70%. The temperature and humidity in the room were monitored using a thermo-hygrometer, and measurements were recorded twice daily (once in the morning and once in the afternoon). Lighting in the animal room was controlled by an electronic timing system, with 12 hours of light and 12 hours of darkness daily (lights on at 7:00 AM and off at 7:00 PM). During the experiment, animals were housed in single cages, each with its own toy. During the experiment, animals had free access to food (rat and mouse growth / breeding chow) and drinking water.

[0181] 2. Animals in each group were subcutaneously injected with a solvent (20 mM sodium citrate buffer, pH=7.0) and the polypeptide of the present invention (10 nmol / kg), with the administration time: 9:30 am, administration frequency: once every 3 days, and administration cycle: 22 days.

[0182] C. Experimental Results The experimental results are shown in Table 10. [Table 10]

[0183] Conclusion: The polypeptide of the present invention showed a significant weight-reducing effect in DIO mice.

[0184] Experimental Example 6: Drug efficacy study in db / db mice - in vivo drug efficacy evaluation A. Experimental Objective The purpose was to study the blood glucose control effect of the polypeptide of the present invention on type II diabetic db / db mice.

[0185] B. Experimental Procedure After arriving at the facility, db / db mice were housed in a strictly environmentally controlled animal room maintained at a temperature of 20–24°C and humidity of 30–70%. The temperature and humidity in the room were monitored using a thermo-hygrometer, and measurements were recorded twice daily (once in the morning and once in the afternoon). Lighting in the animal room was controlled by an electronic timing system, with 12 hours of light and 12 hours of darkness daily (lights on at 7:00 AM and off at 7:00 PM). During the experiment, animals were housed in single cages, each with its own toy. During the experiment, animals had free access to food (rat and mouse growth / breeding chow) and drinking water.

[0186] 2. Animals in each group were subcutaneously injected with a solvent (20 mM sodium citrate buffer, pH 7.0) and the polypeptide of the present invention (15 nmol / kg), with the administration time: 9:30 to 11:00 a.m., and the administration frequency: once a day for four consecutive weeks.

[0187] C. Experimental Results The experimental results are shown in Table 11. [Table 11]

[0188] Conclusion: The polypeptide of the present invention exhibits excellent blood glucose lowering effects in db / db mice. (Supplementary Note) The invention of the present disclosure includes the following aspects. <Item 1>[[]END]] A polypeptide having an array represented by the following formula or a pharmaceutically acceptable salt thereof. Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1-Leu-Asp-Lys- 1 Lys-Ala-Gln- 1 Lys-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (I-1)、 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1 -Leu-Asp- 1 Lys-Lys-Ala- 1 Lys-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (I-2)、 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1 -Leu-Asp-Lys-Lys-Ala-Gln- 1 Lys-Ala-Phe- 1 Lys-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (I-3)、 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1 -Leu-Asp-Lys-Lys-Ala-Gln-Aib- 1 Lys-Phe-Ile- 1 Lys-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (I-4)、 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1 -Leu-Asp-Lys-Lys-Ala-Gln-Aib-Ala-Phe-Ile-Glu- 1 Lys-Leu-Leu- 1 Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (I-5)、 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-X 1 -Leu-Asp-Lys-Lys-Ala-Gln-Aib-Ala-Phe-Ile- 1 [[ID=Z7]]Lys-Tyr-Leu- 1 Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-X 2 (II-6) (However, The structure of Aib is

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

Claims

1. A polypeptide represented by the following formula or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】

2. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier thereof.

3. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the manufacture of a medicament for treating diabetes or obesity.

4. Use of the polypeptide described in claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in claim 2 in the manufacture of a medicament for treating diabetes and obesity.

Images